Electrochemical Properties of Living Tissue
A living tissue functions as an electrical machine, and the matrix of cells comprising the tissue exhibit electrical properties including, but not limited to, the ability to conduct electricity, create electric fields, and function as electrical generators. The primary charge carriers in living organisms are negatively charged electrons, positively charged hydrogen protons, positively charged sodium, potassium, calcium and magnesium ions and negatively charged anions, particularly phosphate ions. (Reviewed in Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from royalrife.com/haltiwanger1).
The body uses the exterior cell membrane, and positively charged mineral ions that are maintained in different concentrations on each side of the cell membrane, to create a cell membrane potential (a voltage difference across the membrane) and a strong electrical field around the cell membrane. (As used herein the term “electrical field” refers to the effect which a charged particle or body exerts on charged particles or bodies situated in the medium surrounding it; i.e., if a negatively charged particle is placed within the electric field of a positively charged particle, there will be an attractive force, while there will be a repulsive force if the charges are alike. The electric field is perpendicular to the magnetic field). This electrical field is a readily available source of energy for cellular activities, such as membrane transport, and the generation of electrical impulses in the brain, nerves, heart and muscles. The storage of electrical charge in the membrane and the generation of an electrical field create a battery function so that the liquid crystal (meaning symmetrically packed) eletroactive intermediates and catalysts can transfer membrane charge to DNA. The body also uses the mitochondrial membrane and positively charged hydrogen ions to create a strong membrane potential across the mitochondrial membrane. Hydrogen ions are maintained in a high concentration on the outside of the mitochondrial membrane by the action of the electron transport chain (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from royalrife.com/haltiwanger1).
Animal cells are organized structures with an internal architecture of cytoskeletal proteins that connects all components of the cell. Cellular components do not randomly float around in the cell but are attached to the cytoskeletal framework and the membranes. Cytoskeletal filaments and tubules form a continuous system that links the cell surface to all organelle structures including passage through the nuclear membrane to the chromosomes. The liquid crystal proteins that compose the cytoskeleton support, stabilize and connect the liquid crystal components of the cell membrane with other cell organelles. The cytoskeletal proteins have multiple roles. They serve as mechanical scaffolds that organize enzymes and water and anchor the cell to structures in the extracellular matrix (ECM) via linkages through the cell membrane, and are dynamic network structures that create a fully integrated electronic that structurally and electronically links and integrates the proteins of the extracellular matrix with the cell organelles. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
The vascular space, filled with blood, is strictly within the confines of arteries, veins, arterioles, veinules, and capillaries. The interstitial space is designated as the cell-poor residual space beyond vascular containment and in between the capillaries and the tissue cells. These spaces create an inflammatory divide. Protective enzymes, such as peroxidases, are only present in functional concentrations in the vascular space. The interstitial space is vulnerable to metabolically produced radicals such as peroxide. Synovial space in joints contains hyaluronic acid and resembles the interstitial space Cartilage, which has a poor blood supply and contains a structural form of hyaluronic acid, also resembles the interstitial space. It is the interstitial space that undergoes allergic edema. Inflammation is usually treated with small molecules that can diffuse into the interstitial space and into sites of inflammation. The use of steroids, for example, increases the local permeability of the sites of inflammation, thereby increasing access to the vascular circulation.
The cytoskeleton is also attached through cell membrane connectors to liquid crystal protein polymers located in the external extracellular matrix (ECM) and to other cells. The liquid crystal protein polymers of the ECM are mostly composed of collagen, elastin, hylauronic acid, and interweaving glycoproteins such as fibronectin. The ECM is a transit area for the passage of nutrients from the bloodstream into the cells, for toxins released by the cells that pass through to the bloodstream, and for migrating immune cells involved in inflammatory reactions that secrete cytokines and other inflammatory mediators. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Biochemically, the ECM is a metabolically and electrically active space that is involved in regulating cell growth control. Cellular components of the ECM are involved in the local production of growth factors, growth inhibitors and cytokines that affect the growth and metabolic activity of tissue/organ cells. (Reichart, L. F., “Extracellular matrix molecules,” In “Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins,” (ed. T. Kreis and R. Vale). Oxford, England: Oxford University Press, pgs. 335-344, 1999). Immune cells such as leukocytes, lymphocytes and macrophages that migrate into the ECM are involved in initiating the removal of damaged cells and in stimulating the growth of new cells.
Cells are electromagnetic in nature, and are capable of generating their own electromagnetic fields and of harnessing external electromagnetic energy of the right wavelength to communicate, control and drive metabolic reactions. Communications in living organisms are accomplished by chemical communication through the circulatory system and energetic communication through the nervous system. A solid state electronic communication system has also been hypothesized to operate in series and in parallel with the nervous system through the liquid crystal protein polymer connective system continuum of the cytoskeleton and extracellular matrix. It has been hypothesized that this continuum of liquid crystal connections function as electronic semiconductors and fiberoptic cables allowing the shunting of charge and associated electronic energy in and out of the cell. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Most molecules in the body are electrical dipoles capable of oscillations and resonance. Electric fields induce or cause alignment of dipole moments. A dipole moment is a function of polarization processes and the strength of the electric field. When biological tissue is exposed to an electric field in the right frequency and amplitude windows, a preferential alignment of dipoles becomes established. Since the cell membrane contains many dipole molecules, an applied electric field causes a preferential alignment of the dipoles. Both internally generated and externally applied electromagnetic fields can affect cell functions. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Electrical Properties of the Cell in Disease Repair and Healing
The body uses electricity (biocurrents) as part of its mechanism for controlling growth and repair. Some of these biocurrents travel through hydrated liquid crystal semiconducting (the term “semiconductor” refers a material whose conductivity lies between that of an electrical conductor, such as a metal, and an insulator) protein-proteoglycan (collagen-hyaluronic acid) complexes of the ECM. It has been hypothesized that biocurrents in the ECM pass through the cell membrane into the cell and electrons produced in the cell also pass out through the cell membrane. The biological liquid crystal molecules and structures such as hyaluronic acid, prothrombin, DNA, cytoskeletal proteins and cell membranes maintain both an inward and outward current. The inward current flows from the cell membrane to cell structures like mitochondria and DNA, and the outward current flows back along liquid crystal semiconducting cytoskeletal proteins through the cell membrane to the ECM. Electrical charges stored in the cell membrane (capacitance) and electrical charges of oxygen free radicals are normally transferred to DNA and are involved in DNA activation and the creation of an electrical field around DNA. DNA is very effective in transferring large amounts of electrical charge along its long axis. (Haltiwanger (2010) citing Garnett M., “First Pulse: A Personal Journey in Cancer Research,” New York, N.Y.: First Pulse Projects, 1998). An alternating current oscillating circuit between the cell membrane and DNA conducted over the electronic liquid crystal network of the cell is thought to be involved in cellular processes such as gene expression. (Garnett, M. and Remo, J. L., “DNA Reductase: A Synthetic Enzyme with Opportunistic Clinical Activity Against Radiation Sickness,” International Symposium on Applications of Enzymes in Chemical and Biological Defense, Orlando, Fla., May, 2001, p. 41.)
It has also been hypothesized that electrical pathways between the cell membrane and DNA are related to cell development, and use anaerobic mechanisms of ATP production. This natural electrical pathway is thought to be transiently disrupted in healthy cells that are involved in wound healing and inflammation, and permanently disrupted in cancer cells that rely on anerobic glycolysis for energy production. (Haltiwanger (2010) citing Garnett M., “First Pulse: A Personal Journey in Cancer Research,” New York, N.Y.: First Pulse Projects, 1998).
Glycoproteins secreted from the cell interior and cellular components of the ECM produce a glycocalyx that covers the cells. These glycoproteins characteristically have a negative charge. The negative charges of the ECM-glycocalyx interface help determine water balance, ion balance and osmotic balance both in the ground substance of the ECM and inside the cells. ECM proteoglycans exist in fern shapes that allow electric charges to flow, and in disorganized shapes that impair transit through the ECM of electrical currents and nutrients. These disorganized shapes occur in the presence of tissue inflammation and toxins, such as free radicals, reactive oxygen species (e.g. superoxide, peroxide, or hydroxyl ions) in the ECM. Such structures produce pockets of high electrical resistance. (Haltiwanger (2010)).
Measuring Electrical Properties of Biological Tissue
The electrical properties of biological tissue can be measured when current flows through the tissue by a phenomenon termed “impedance” or alternatively “bioimpedance”, which refers to the opposition to the flow of alternating current through a conductor, and is described by a relation between voltage and current in a system. (Holder, D. S., “Appendix A: A brief introduction to bioimpedance,” in “Electrical Impedance Tomography”, Institute of Physics Publishing, Bristol and Philadelphia (2005), pp. 411-422). Impedance is defined as the ratio of incremental change in voltage to the resulting current (or vice versa) across an electrochemical cell or an electrical circuit. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)).
Impedance can be measured in tissues and cells using electrochemical impedance spectroscopy (EIS). Through the application of a small sinusoidally varying potential U, one measures the resulting current response I. By repeating the process at varying excitation frequencies f, impedance can be calculated as a function of the angular frequency ω, given by the relation:
            Z      ⁡              (                  j          ⁢                                          ⁢          ω                )              =                            U          ⁡                      (            jω            )                                    I          ⁡                      (                          j              ⁢                                                          ⁢              ω                        )                              =                                    Z            r                    ⁡                      (            ω            )                          +                  j          ⁢                                          ⁢                                    Z              i                        ⁡                          (              ω              )                                            ,where ω=2Πf. (Grieshaber, D. et al., “Electrochemical biosensors,” Sensors, 8: 1400-1458 (2008)).
More specifically, when applying a sinusoidal voltage reference Vref(t)=|Vref|sin(ω0t) across the cell and assuming linear behavior, the corresponding current flowing through the cell is I(t)=|I|sin(ω0t+θ), wherein θ is the phase shift of the signal with respect to the excitation. Thus, the relationship between excitation and readout signals depends only on phases and amplitude ratios. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)). Therefore, impedance is made of two components: resistance or the real part of the data, and reactance, the out-of-phase data. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)).
Resistance (R) is a measure of the extent to which a substance opposes the flow of electrons or, in aqueous solution as in living tissue, the flow of ions among its cells. The three fundamental properties governing the flow of electricity are “voltage”, “current” and “resistance”. Voltage is the pressure exerted on a stream of charged particles moving down a wire or through an ionized salt solution. Current is the amount of charge flowing per unit time. Resistance is the ease or difficulty with which the charged particles can flow. Voltage, current and resistance are related by Ohm's law: V (voltage, Volts)=I (current, Amps)×R (resistance, Ohms (Ω)). Ohm's law applies to both direct current (d.c. or steadily flowing) or alternating current (a.c. or current that flows backwards and forwards).
Capacitance (C) refers to the extent to which an electronic component, circuit, or system stores and releases energy as the current and voltage fluctuate with each AC cycle. The capacitance physically corresponds to the ability of plates in a capacitor to store charge. With each cycle, charges accumulate and then discharge. Direct current cannot pass through a capacitor. Alternating current can pass because of the rapidly reversing flux of charge. The capacitance is an unvarying property of a capacitive or more complex circuit. However, the effect in terms of the ease of current passage depends on the frequency of the applied current; charges pass backwards and forwards more rapidly if the applied frequency is higher.
Reactance (X), analogous to resistance, refers to the current travelling through a capacitor or a coil. A higher reactance has a higher effective resistance to alternating current. Like resistance, its value is in Ohms, but it depends on the applied frequency, and is described by the relation: Reactance (Ohms)=1/(2×Π×Frequency (Hz)×Capacitance (Farads)). When a current is passing through a purely resistive circuit, the voltage recorded across the resistor will coincide exactly with the timing, or phase, of the applied alternating current. However, when current flows across a capacitor, the voltage recorded across it lags behind the applied current because of back and forth flow of current requiring alternating charging and discharging of the plates of the capacitor. In terms of a sine wave which has 360° in a full cycle, the lag is one quarter of a cycle, i.e., 90°.
Impedance is the frequency dependent resistance derived from the following three components of an AC circuit: direct current (DC) resistance; capacitive resistance; and inductive reactance. Capacitance is produced by storing charge on a surface at an energy expense producing a retardation of voltage flow. Inductance is produced by storing energy in a magnetic field in bulk space at an energy expense producing a retardation of current flow. Capacitance is counted in Farad units and inductance in Henry units. These two retardation effects are combined in a process and representation called the phase angle, which is the angular summation of the two waves or pulses of voltage and current. In the Mott-Schottky form of impedance measurement, only a single frequency influence is used. This is a departure from Nyquist or Cole (Cole, K. S, and Cole, R. H, “Dispersion and Absorption in Dielectrics. I. Alternating Current Characteristics,” J. Chem. Phys. 9: 341-351 (1941)) plots, which utilize a descending frequency series. The Mott-Schottky method is useful for analysis of the underlying impedance vectors within devices and within molecules.
Palladium Compounds and Complexes
Certain palladium compounds have been described as inhibitors of growth, and have been shown to be interactive, or able to bind, with DNA. Such working concepts of growth inhibition are quite general and the mechanism of disease specificity has not been further approached.
Palladium is a transition metal of Group 10 of the periodic table. Its electron configuration is 1s22s2p6, 3s2p6d10, 4s2p6d10. Palladium is a well-known catalyst. The d-orbital contribution of palladium allows an exaggerated extension of electronic radii, thereby minimizing Coloumbic attractive force and giving the d-orbital electrons the properties of unpaired electrons. Consequently, palladium presents a stable electronic state closely resembling the free radical state.
According to quantum theory, each electron has a spin that is associated with an angular momentum leading to a magnetic moment. Consequently, the negative charge carried by the electron is also associated with a spin resulting in a circulating electric current. The circulating current induces a magnetic moment μS which, if the electron is subjected to a steady magnetic field H0, causes the electron to experience a torque that tends to align the magnetic moment with the field. The energy of the system depends upon the projection of the spin vector along H0. Quantum theory stipulates that only two values are permitted for an electron, which means that the electron magnetic moment can only assume two projections or spin states onto the applied field: the “+½ spin state”, when the electron's magnetic moment μS is aligned with the direction of the applied magnetic field H0; and the “−½ spin state”, when the electron's magnetic moment μS is aligned opposed to the direction of H0. Consequently, the ensemble of energy levels also reduces to two values, designated as E+, a lower energy level corresponding to the +½ spin state (aligned with the direction of the applied magnetic field) and E−, a higher energy level corresponding to the −½ spin state (opposed to the direction of the applied magnetic field). Because the +½ spin state is of slightly lower energy, in a large population of electrons, slightly more than half of the electrons will occupy this state, while slightly less than half will occupy the −½ spin state. The slight excess of the electron spin in the direction of the magnetic field constitutes a slight net magnetization of the material, a phenomenon known as spin polarization. The difference in energy between the two spin states increases with increasing strength of the magnetic field H0. The higher the strength of H0, the more the net magnetization or the spin polarization, i.e. the higher the number of electrons that will occupy the +½ state as compared to the −½ state.
In addition to the spinning motion, the angular momentum vector of a spinning electron as a result of the torque exhibits a precession around the external field axis with an angular frequency ωL. The precessional motion, known as Larmour precession, is similar to a spinning top whose spin axis rotates slowly around the vertical. The frequency of precession, ωL termed the Larmour frequency, is the number of times per second the electron precesses in a complete circle. The precessional frequency increases the strength of the magnetic field H0.
If an electron that is precessing in an applied magnetic field is exposed to electromagnetic radiation of a frequency ωA that matches with the precessional frequency ωL, the resulting condition is known as resonance. In the resonance condition, an electron of a lower energy +½ spin state (aligned with the applied magnetic field) will transition or flip to the higher energy −½ spin state (opposed to the applied magnetic field). In doing so, the electron absorbs radiation at this resonance frequency, ωA=ωL. This frequency corresponds to the separation between the energy levels of the two spin states, equal to ΔE=E+−E−. This phenomenon is called electron spin resonance (ESR). ESR measures a molecular splitting constant, which is the Gaussian distance or hyperfine shift between the repetitive peaks.
When stimulated by a reaction, the rate of precession can increase, and the dynamic effect is described as Rabi frequency. According to Maxwell-Faraday-Heaviside laws, a moving charge produces a magnetic field in its path, given by: Curl B=4 pi C, where Curl is the net circulating magnetic energy, and C is the charge density or rate of charge moving through a cross section of space or material.
When an atom or molecule has an even number of electrons, electron spins pair off in atomic or molecular orbitals so that virtually no net spin magnetism is exhibited; such material is said to be “diamagnetic”. However, when an atom or molecule has an odd number of electrons, complete pairing is not possible and the material is said to be “paramagnetic”. The phenomena of spin magnetism (spin polarization) and ESR are observed in paramagnetic materials. The minimally attracted spinning d-orbital electrons in palladium render paramagnetic properties in palladium complexes.
The motion of the palladium d-orbital electrons produces an intermittent or pulsed magnetic field. In suitable palladium complexes, the d-orbital electrons are capable of introducing long range molecular magnetic signals into chemical systems. Thus, palladium can form coordination complexes with suitable solubility, voltametric behavior, and oxidation state, such as the palladium-lipoic acid complex (PdLA). The transfer of electrons from PdLA to DNA or RNA and their consequent interactions have been shown with voltammetry and ESR spectroscopy. The shunting of electron energy from PdLA to DNA or RNA has been hypothesized to alter the nucleic acid configuration to heterochromatin. (U.S. Pat. Nos. 5,463,093, 5,679,697 and 5,776,973).
Palladium lipoic acid (PdLA) complexes and their use in the treatment of tumors and psoriasis have been disclosed in U.S. Pat. Nos. 5,463,093, 5,679,697 and 5,776,973, each of which is incorporated by reference in its entirety. Crystallographic studies have shown that the palladium lipoic acid complex forms a trigonal prism. The bonds of the palladium-lipoic acid complex are coordinate covalent, with the complex between palladium and lipoic acid bonded (1) at the carbonyl of the carboxyl group with probable resonance involvement of both oxygens; and (2) at one or more sulfur atoms. The result is a bent chain of lipoic acid, with its ends bonded by way of palladium coordination.
PdLA is a charge transfer catalyst and a synthetic DNA reductase. It was shown to electronically reduce DNA by spin coupling to the guanine base and to thereby condense chromatin to the heterochromatic inactive state. (Garnett, M., Krishnan, C., Jones, B., Spin coupled DNA, Electrochem. Soc. 217th Meeting, A774, 2010). Microscopic effects of PdLA on tumor cells and on yeast showed that cell nuclei were condensed by PdLA into a heterochromatic configuration that is associated with gene silencing. (U.S. Pat. Nos. 5,463,093, 5,679,697 and 5,776,973). It has recently been shown that regional mutation-rate variation is strongly associated with regional variation in chromatin organization into heterochromatin- and euchromatin-like domains. (Schuster-Buchler, B and Lehner, B., Nature 488: 504-507 (2012). PdLA was shown to resemble fern structures of liquid crystal polymers of ECM. (Garnett, M. and Remo, J. L., “DNA Reductase: A Synthetic Enzyme with Opportunistic Clinical Activity Against Radiation Sickness,” International Symposium on Applications of Enzymes in Chemical and Biological Defense, Orlando, Fla., May, 2001, p. 41.)
Palladium and lipoic acid have unique electronic properties. In vivo, lipoic acid is part of mitochondrial Complex I, pyruvic dehydrogenase, and therefore directly interacts with the charge relay system of the mitochondria as a source of charge.
In parallel research, organo-palladium-zinc complexes of general formula (Me)a(Lipoic acid)b(fatty acid)c(amino acid)e, where a, b and c are each 1, d is 0 or 1, and e is 0, 1, or, wherein the standard potential of the complex is electropositive (meaning having a positive electric charge tending to attract electrons), have been developed. See PCT/US2011/032114, filed on Apr. 12, 2011, entitled NOVEL ORGANO-PALLADIUM COMPLEXES, which is incorporated by reference in its entirety. These complexes are cytotoxic to breast cancer, brain cancer and Ehrlich ascites carcinoma cells.
The inorganic catalyst literature identifies the palladium-ruthenium system (Pd—Ru) (Tripathi, S. N., Bharadwaj, S. R., Dharwadkar, S. R., The Pd—Ru System (Palladium-Ruthenium), J. of Phase Equilibria, V. 14, No. 5, 638-642, 1993; Adams, R. D., Captain, B. F. W., Smith, M. D. Lewis Acid-Base Interactions Between Metal Atoms and their Applications for the Synthesis of Bimetallic Cluster Complexes, J. Am. Chem. Soc., V. 124, No. 20, 5628-9, May 2002) as having a singular peritectic phase with synergic effect on the catalytic hydrogenation of nitroaromatics (Wan, B. S., Liao, S. J., XU, Y., Yu, D. R., Synergic Effect of Palladium-Based Bimetallic Catalysts for the Hydrogenation of Nitroaromatics, Reaction Kinetics and Catalysis Letters, V. 63, No. 2, 397-401). In peritectic transformations, a liquid and solid of fixed proportions react to form a new microcrystal phase capable of nucleation and growth. Pd—Ru also has Lewis acid-base interactions between the metal atoms (Tripathi, S. N., Bharadwaj, S. R., Dharwadkar, S. R., The Pd—Ru System (Palladium-Ruthenium), J. of Phase Equilibria, V. 14, No. 5, 638-642, 1993; Adams, R. D., Captain, B. F. W., Smith, M. D. Lewis Acid-Base Interactions Between Metal Atoms and their Applications for the Synthesis of Bimetallic Cluster Complexes, J. Am. Chem. Soc., V. 124, No. 20, 5628-9, May 2002). Ruthenium is a transition metal of group 8 of the periodic table. Its electronic configuration is 1s2 2s2p6 3s2p6d10 4s2p6d7 5s1.
The present invention describes organo-metallic complexes comprising palladium, ruthenium, and zinc, and their charge transfer properties.